First Report of root rot and wilt caused by Pythium myriotylum on hemp (Cannabis sativa L.) in the United States

In September 2018, a hemp plant (Cannabis sativa L. cv. Dinamed) presented severe wilting, root rot, and mortality in a research greenhouse at the University of Connecticut in Storrs, CT. The hemp plant was grown in a 11.36-liter container with soilless peat-based potting mix (SunGro Fafard 3B, Agawam, MA, U.S.A.). The plants were in a polycarbonate greenhouse with a daytime heating set point of 20°C and a ventilation set point of 26°C under 18 h of supplemental lighting. Roots were collected from the symptomatic hemp plant, washed three times with sterile deionized water, blotted dry, and plated on PARP selective medium (Jeffers and Martin 1986). The plates were incubated in the dark at 21°C for 48 h. Mycelia resembling the morphology of Pythium species were observed in all plates. Following van der Plaats-Niterink’s key (van der Plaats-Niterink 1981), the isolate was identified as Pythium myriotylum based on the morphology of sexual and asexual structures. Colonies grown on sterile tall fescue leaves had filamentous, inflated, nonproliferating sporangia that ranged in width between 6 and 16 μm. Oogonia were 28 to 30 μm in diameter, globose, and had a smooth, thick wall (1 to 2 µm). The antheridia were curved, bulbous, and developed on branched stalks with multiple antheridia per oogonium. The appressoria were clavate, and oospores were mostly aplerotic. DNA was extracted from mycelial mats using the DNeasy Plant Mini Kit (Qiagen, Germantown, MD, U.S.A.). The cytochrome oxidase subunits I and II (coxI and coxII) (Martin 2000; Robideau et al. 2011) and the internal transcribed spacer (ITS) regions 1 and 4 (White et al. 1990) were amplified. The amplicons were sequenced and BLAST analyzed in NCBI GenBank. The isolate was confirmed to be P. myriotylum with ≥98% query coverage and 100% identity match to GenBank accession numbers LC440564.1, KJ595365.1, and KX671106. The nucleotide sequences obtained in this project have been assigned the GenBank accession numbers MN037880, MN037881, and MN022496 for coxI, coxII, and ITS, respectively. To confirm Koch’s postulates, three 68-day-old hemp plants (cv. Wife) were inoculated with P. myriotylum with 1 × 10⁵ oospores/ml obtained from three mycelial mats per pot. The mycelial mats, grown in V8 broth, were blended with 500 ml of deionized water, oospores were quantified using a hemocytometer, and the inoculum was applied directly in the potting medium so that no leaching occurred. The hemp plants were grown in 11.36-liter containers in a growth chamber set at 24°C with 65% humidity under 16 h of light. After 20 days, two of the three inoculated plants were wilted, and the control plants were symptomless. Necrotic roots were collected from all the plants. Ten root fragments were plated on PARP selective medium (replicated four times, one plate per replicate). The mycelial growth emanating from roots resembled Pythium on isolation plates from the symptomatic plants, and no growth was observed from roots of noninoculated plants or the asymptomatic plant. Colonies isolated from the root tissue morphologically resembled the originally isolated organism, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of P. myriotylum being detected on a hemp plant in the United States.